Table 1 Summary of αSyn-SAA reagents and protocols for each laboratory

Monomeric αSyn

0.1 mg/ml recombinant human αSyn (w/o tag)

0.3 mg/ml recombinant αSyn (C-term His)

0.1 mg/ml recombinant K23Q αSyn (N-term His)

CSF

5% v/v

20% v/v

15% v/v

Reaction volume

100 μl

200 μl

100 μl

Reaction duration

70 h

150 h

48 h

Reaction mixture

100 mM NaH₂PO₄

(pH to 8.2 w/ NaOH)

500 mM NaCl

100 mM PIPES

(pH to 6.5 w/ NaOH)

170 mM NaCl

40 mM NaH₂PO₄/ Na₂HPO₄

(pH to 8.0 w/ Na₂HPO₄)

0.0015% SDS

Temperature

33 °C

37 °C

42 °C

Beads

25 ± 3 mg of 0.10-mm zirconia/silica beads

Single 3/32-inch Si₃N₄ bead

0.8-mm silica beads (6)

Shaking protocol

200 rpm shaking for 60 s and 14 min rest

700 rpm shaking for 60 s every 30 min

400 rpm shaking for 60 s every 60 s

No. Replicates

8

3

4

Threshold (RFU)

3500

Probabilistic algorithm based on assay kinetic parameters

10% plate maximum fluorescence

Positive

 ≥ 50% positive replicates (4–8)

All 3 replicates positive by algorithm

 > 25% replicates positive

Negative

 < 50% positive replicates (0–3)

0–1 positive replicates

0 positive replicates

Inconclusive

n/a

2 positive replicates

if 25% replicates positive, retested